The present invention relates generally to the identification of a novel member of the type I interferon family. More specifically, the present invention concerns the isolation of a novel nucleic acid encoding a new and distinct type I interferon, termed interferon-epsilon (IFN-xcex5).
Interferons are relatively small, single-chain glycoproteins released by cells invaded by viruses or certain other substances. Interferons are presently grouped into three major classes, designated leukocyte interferon (interferon-alpha, xcex1-interferon, IFN-xcex1), fibroblast interferon (interferon-beta, xcex2-interferon, IFN-xcex2), and immune interferon (interferon-gamma, xcex3-interferon, IFN-xcex3). In response to viral infection, lymphocytes synthesize primarily xcex1-interferon (along with a lesser amount of a distinct interferon species, commonly referred to as omega interferon, IFN-xcfx89), while infection of fibroblasts usually induces xcex2-interferon. xcex1- and xcex2-interferons share about 20-30 percent amino acid sequence homology. Thus, the gene for human IFN-xcex2 lacks introns, and encodes a protein possessing 29% amino acid sequence identity with human IFN-xcex1I, suggesting that IFN-xcex1 and IFN-xcex2 genes have evolved from a common ancestor (Taniguchi et al., Nature 285, 547-549 (1980)). By contrast, IFN-xcex3 is not induced by viral infection, rather, is synthesized by lymphocytes in response to mitogens, and is scarcely related to the other two types of interferons in amino acid sequence. Interferons-xcex1, xcex2 and xcfx89 are known to induce MHC Class I antigens, and are referred to as type I interferons, while IFN-xcex3 induces MHC Class II antigen expression, and is also referred to as type II interferon.
A large number of distinct genes encoding different species of IFNs-xcex1 have been identified. Alpha interferon species identified previously fall into two major classes, I and II, each containing a plurality of discrete proteins (Baron et al., Critical Reviews in Biotechnology 10, 1790190 (1990); Nagata et al., Nature 287, 401-408 (1980); Nagata et al., Nature 284, 316-320 (1980); Streuli et al., Science 209, 1343-1347 (1980); Goeddel et al., Nature 290, 20-26 (1981); Lawn et al., Science 212, 1159-1162 (1981); Ullrich et al., J. Mol. Biol. 156, 467-486 (1982); Weissmann et al., Phil. Trans. R. Soc. Lond. B299, 7-28 (1982); Lund et al., Proc. Natl. Acad. Sci. 81, 2435-2439 (1984); Capon et al., Mol. Cell. Biol. 5, 768 (1985)). The various IFN-xcex1 species include IFN-xcex1A (IFN-xcex12), IFN-xcex1B, IFN-xcex1C, IFN-xcex1C1, IFN-xcex1D (IFN-xcex11), IFN-xcex1E, IFN-xcex1F, IFN-xcex1G, IFN-xcex1H, IFN-xcex1I, IFN-xcex1J1, IFN-xcex1J2, IFN-xcex1K, IFN-xcex1L, IFN-xcex14B, IFN-xcex15, IFN-xcex16, IFN-xcex174, IFN-xcex176 IFN-xcex14a), IFN-xcex188, and alleles of these species. According to our current knowledge, the IFN-xcex1 family consists of 13 expressed alleles producing 12 different proteins that exhibit remarkably different biological activity profiles. Pestka, S., Semin. Oncol. 24(suppl. 9), S9-4-S9-17 (1997).
Interestingly, while only a single human IFN-xcex2 gene has been unequivocally identified, bovine IFN-xcex2 is encoded by a family of five or more homologous, yet distinct genes.
Interferons were originally produced from natural sources, such as buffy coat leukocytes and fibroblast cells, optionally using known inducing agents to increase interferon production. Interferons have also been produced by recombinant DNA technology.
The cloning and expression of recombinant IFN-xcex1A (rIFN-xcex1A, also known as IFN-xcex12) was described by Goeddel et al., Nature 287, 411 (1980). The amino acid sequences of rIFNs-xcex1A, B, C, D, F, G, H, K and L, along with the encoding nucleotide sequences, are described by Pestka in Archiv. Biochem. Biophys. 221, 1 (1983). The amino acid sequences and the underlying nucleotide sequences of rIFNs-xcex1E, I and J are described in British Patent Specification No. 2,079,291, published Jan. 20, 1982. Hybrids of various IFNs-xcex1 are also known, and are disclosed, e.g. by Pestka et al., supra. Nagata et al., Nature 284, 316 (1980), described the expression of an IFN-xcex1 gene, which encoded a polypeptide (in non-mature form) that differs from rIFN-xcex1D by a single amino acid at position 114. Similarly, the cloning and expression of an IFN-xcex1 gene (designated as rIFN-xcex12) yielding a polypeptide differing from rIFN-xcex1A by a single amino acid at position 23, was described in European Patent Application No. 32 134, published Jul. 15, 1981.
The cloning and expression of mature rIFN-xcex2 is described by Goeddel et al., Nucleic Acids Res. 8, 4057 (1980).
The cloning and expression of mature rIFN-xcex3 are described by Gray et al., Nature 295, 503 (1982).
IFN-xcfx89 has been described by Capon et al, Mol. Cell. Biol. 5, 768 (1985).
IFN-xcfx84 has been identified and disclosed by Whaley et al., J. Biol. Chem. 269, 10864-8 (1994).
All of the known IFNs-xcex1, -xcex2, and -xcex3 contain multiple cysteine residues. These residues contain sulfhydryl side-chains which are capable of forming intermolecular disulfide bonds. For example, the amino acid sequence of mature recombinant rIFN-xcex1A contains cysteine residues at positions 1, 29, 98 and 138. Wetzel et al., Nature 289, 606 (1981), assigned intramolecular disulfide bonds between the cysteine residues at positions 1 and 98, and between the cysteine residues at positions 29 and 138.
Antibodies specifically binding various interferons are also well known in the art. For example, anti-xcex1-interferon agonist antibodies have been reported by Tsukui et al., Microbiol. Immunol. 30,112901139 (1986); Duarte et al., Interferon-Biotechnol. 4, 221-232 (1987); Barasoaian et al., J. Immunol. 143 507-512 (1989); Exley et al., J. Gen. Virol. 65, 2277-2280 (1984); Shearer et al, J. Immunol. 133, 3096-3101 (1984); Alkan et al., Ciba Geigy Foundation Symposium 119, 264-278 (1986); Noll et al., Biomed. Biochim. Acta 48, 165-176 (1989); Hertzog et al., J. Interferon Res. 10(Suppl. 1) (1990); Kontsek et al., J. Interferon Res. (special issue) 73-82 (1991), and U.S. Pat. No. 4,423,147 issued Dec. 27, 1983.
The actions of type I interferons appear to be mediated by binding to the IFN-xcex1 receptor complex on the cell surface. This receptor is composed of at least two distinct subunits identified as IFN-xcex1tR1 (Uze et al., Cell 60, 225-234 [1990]) and IFN-xcex1R2 (Novick et al., Cell 77 391-400 [1994]), each having 2 and 3 spliced variants, respectively. IFN-xcex1R2 is the binding subunit of the known type I interferons, whereas IFN-xcex1R1 contributes to higher affinity binding and signaling. The engagement of receptors by ligand binding activates Janus family kinases (JAK) and protoplasmic latent signal transducers and activators of transcription (STAT) proteins by tyrosine phosphorylation. Activated STATs translocate to the nucleus in forms of complexes and interact with their cognitive enhancer elements of IFN-stimulated genes (ISGs), leading to a corresponding transcription activation and biological responses. Darnell et al., Science 264, 1415-21 (1994). However, despite similarities in their binding properties, the biological responses stimulated by type I interferons are significantly different.
Interferons have a variety of biological activities, including antiviral, immunoregulatory and antiproliferative properties, and are, therefore, of great interest as therapeutic agents in the control of cancer, and various viral diseases. Interferons have been implicated in the pathogenesis of various autoimmune diseases, such as systemic lupus erythematoses, Behget""s disease, insulin-dependent diabetes mellitus (IDDM, also referred to as type I diabetes). It has been demonstrated in a transgenic mouse model that xcex2 cell expression of IFN-xcex1 can cause insulitis and IDDM, and IFN-xcex1 antagonists (including antibodies) have been proposed for the treatment of IDDM (WO 93/04699, published Mar. 18, 1993). Impaired IFN-xcex3 and IFN-xcex1 production has been observed in multiple sclerosis (MP) patients. An acid-labile IFN-xcex1 has been detected in the serum of many AIDS patients, and it has been reported that the production of IFN-xcex3 is greatly suppressed in suspensions of mitogen-stimulated mononuclear cells derived from AIDS patients. For a review see, for example, Chapter 16, xe2x80x9cThe Presence and Possible Pathogenic Role of Interferons in Diseasexe2x80x9d, In: Interferons and other Regulatory Cytokines, Edward de Maeyer (1988, John Wilet and Sons publishers). Alpha and beta interferons have been used in the treatment of the acute viral disease herpes zoster (T. C. Merigan et al., N. Engl. J. Med. 298, 981-987 (1978); E. Heidemann et al., Onkologie 7, 210-212 (1984)), chronic viral infections, e.g. hepatitis B infections (R. L. Knobler et al., Neurology 34, 1273078 (1984); M. A. Faerkkilae et al., Act. Neurol. Sci. 69 184-185 (1985)). rIFN-xcex1-2a (ROFERON(copyright), Roche) is an injection formulation indicated in use for the treatment of hairy cell leukemia and AIDS-related Kaposi""s sarcoma. Recombinant IFN-xcex1-2b (INTRON(copyright) A, Schering) has been approved for the treatment of hairy cell leukemia, selected cases of condylomata acuminata, AIDS-related Kaposi""s sarcoma, chronic hepatitis Non-xcex1, Non-B/C, and chronic helatitis B infections is certain patients. IFN-xcex3-1b (ACTIMMUNE(copyright), Genentech, Inc.) is commercially available for the treatment of chronic granulomatous disease.
For further information about the biologic activities of type I IFNs see, for example, Pfeffer, Semin. Oncol. 24(suppl 9), S9-63-S9-69 (1997).
Applicants have identified a cDNA clone (designated in the present application as xe2x80x9cDNA50960xe2x80x9d) that encodes a novel human interferon polypeptide, which is now designated as human IFN-xcex5.
In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA having at least a 95% sequence identity to (a) a DNA molecule encoding a novel human interferon polypeptide originally designated PRO655, and hereinafter also referred to as IFN-xcex5, comprising the sequence of amino acids from about 22 to 189 of FIG. 1 (SEQ ID NO: 1), or (b) the complement of the DNA molecule of (a). In one aspect, the isolated nucleic acid comprises DNA encoding a new interferon polypeptide having at least amino acid residues 22 to 189 of FIG. 1 (SEQ ID NO:1), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another embodiment, the isolated nucleic acid molecule encodes the full-length polypeptide represented in FIG. 1 (SEQ. ID. NO:1), with or without the putative signal peptide at amino acids 1-21, and with or without the initiating methionine, or is the complement of such DNA molecule. In a further embodiment, the isolated nucleic acid molecule comprises DNA having at least a 95% sequence identity to (a) DNA molecule encoding the same mature polypeptide encoded by the human interferon protein cDNA in ATCC Deposit No.209509 (DNA50960-1224), deposited on Dec. 3, 1997.
In another embodiment, the invention provides a vector comprising DNA (as hereinabove defined) encoding a novel interferon-xcex5 polypeptide. A host cell comprising such a vector is also provided. By way of example, the host cells may be CHO cells, E. coli, or yeast (including Saccharomyces cerevisiae and other yeast strains). A process for producing the new interferon polypeptides of the present invention is further provided and comprises culturing host cells under conditions suitable for expression of the desired interferon polypeptide, and recovering the interferon from the cell culture.
In another embodiment, the invention provides novel, isolated interferon-xcex5 polypeptides. In particular, the invention provides isolated a native interferon-xcex5 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 22 to 189 of FIG. 1 (SEQ ID NO:1). In another embodiment, the IFN-xcex5 polypeptide has at least about 95% sequence identity with the native human IFN-xcex5 polypeptide specifically disclosed in 25 the present application, and preferably retains the pair of cysteine residues at amino acid positions 32 and 142. Both glycosylated and unglycosylated forms of the IFN-xcex5 polypeptides are included.
In another embodiment, the invention provides chimeric molecules comprising a novel interferon-xcex5 polypeptide herein fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises an interferon-xcex5 polypeptide fused to an epitope tag sequence or an immunoglobulin heavy or light chain constant region sequence, e.g. the Fc region of an immunoglobulin.
In another embodiment, the invention provides an antibody which specifically binds to a novel interferon-xcex5 polypeptide disclosed herein Optionally, the antibody is a monoclonal antibody.
In a further aspect, the present invention concerns compositions comprising an effective amount of an IFN-xcex5 polypeptide, or an agonist thereof, in admixture with a pharmaceutically acceptable carrier. The composition may, for example, be used for the inhibition of neoplastic cell growth, e.g. for the treatment of various tumors, including cancers, such as leukemias, AIDS-related Kaposi""s sarcoma, etc. In a particular embodiment, the composition comprises a cytostatic amount of an IFN-xcex5 polypeptide, or an agonist thereof. In a preferred embodiment, the composition comprises a growth inhibitory amount of an IFN-xcex5 polypeptide, or an agonist thereof. In another preferred embodiment, the composition comprises a cytotoxic amount of an IFN-xcex5 polypeptide, or an agonist thereof. In yet another preferred embodiment, the composition comprises IFN-xcex5 in an amount capable of evoking apoptosis of a target cell. Optionally, the compositions may contain one or more additional growth inhibitory and/or cytotoxic and/or other chemotherapeutic agents. In a further embodiment, the compositions may be used to treat viral infections, such as, the acute viral disease zoster, chronic viral infections, e.g. chronic hepatitis non-A, non-B and chronic hepatitis B infections, etc. In a still further embodiment, the compositions are used to upregulate the immune system.
In another aspect, the invention concerns a method for inhibiting the growth of a tumor cell comprising exposing the cell to an effective amount of an IFN-xcex5 polypeptide, or an agonist thereof. In a particular embodiment, the agonist is an anti-IFN-xcex5 agonist antibody. In another embodiment, the agonist is a small molecule that mimics the biological activity of a native IFN-xcex5 polypeptide. The treatment may be performed in vitro or in vivo.
In yet another aspect, the invention concerns a method for treating a viral infection comprising administering a therapeutically effective amount of an IFN-xcex5 polypeptide, or an agonist thereof.
In a further aspect, the invention concerns a method for upregulation of the immune system comprising administering a therapeutically effective amount of an IFN-xcex5 polypeptide, or an agonist thereof.
In a still further embodiment, the invention concerns an article of manufacture, comprising:
a container; and
a composition comprising an active agent contained within the container; wherein the composition is effective for inhibiting neoplastic cell growth, e.g. growth of tumor cells, and/or to cause apoptosis of such cells, and the active agent in the composition is an IFN-xcex5 polypeptide, or an agonist thereof. In a particular embodiment, the agonist is an anti-IFN-xcex5 agonist antibody. In another embodiment, the agonist is a small molecule that mimics the biological activity of a native IFN-xcex5 polypeptide.
Similarly, articles of manufacture comprising IFN-xcex5 in an amount effective to treat viral infections and/or to upregulate the immune system are within the scope of the invention.
In a further embodiment, the invention concerns a method for screening compounds for anti-tumor activity. In one aspect, the screening assay is designed to identify agonists of a native IFN-xcex5 polypeptide by testing the ability of a candidate compound to inhibit the growth of a tumor cell the growth of which has been inhibited by a native IFN-xcex5 polypeptide, or a fragment thereof. In another embodiment, the screening assay is designed to identify compounds that are capable of enhancing the expression level of a native IFN-xcex5 polypeptide in a biological cell sample in which the expression of level of the native protein has been determined to be subnormal.
In yet another embodiment, the invention concerns a method for the prognosis or diagnosis of tumor in a mammal, comprising determining in a test sample taken from the mammal, the expression level of an IFN-xcex5 polypeptide, and comparing the result with the expression level of the same polypeptide in a test sample taken from a healthy mammal of the same species, under identical conditions. Subnormal expression of any of the IFN-xcex5 gene may be indicative that the mammal tested has a tendency to develop a tumor, or has already developed tumor.
The invention further concerns compositions comprising an effective amount of an IFN-xcex5 antagonist, e.g. an antagonist anti-IFN-xcex5 antibody or a small molecule antagonist. Such compositions may be used for the treatment of conditions associated with the overexpression of IFN-xcex5. Without limitation, such conditions include autoimmune diseases, such as systemic lupus erythematoses, Behxc3xa7et""s disease, and insulin-dependent diabetes mellitus (IDDM, also referred to as type I diabetes). Methods for treating such conditions are also within the scope of the invention.